1. Technical Field
The present invention pertains generally to viral proteins. In particular, the invention relates to improved methods for isolating truncated forms of hepatitis C virus E1 and E2 proteins having improved biological properties for use in vaccine compositions and as diagnostic reagents.
2. Background of the Invention
Hepatitis C Virus (HCV) is the principal cause of parenteral non-A, non-B hepatitis which is transmitted largely through blood transfusion and sexual contact. The virus is present in 0.4 to 2.0% of blood donors. Chronic hepatitis develops in about 50% of infections and of these, approximately 20% of infected individuals develop liver cirrhosis which sometimes leads to hepatocellular carcinoma. Accordingly, the study and control of the disease is of medical importance.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, there are three putative structural proteins, consisting of the N-terminal nucleocapsid protein (termed “core”) and two envelope glycoproteins, “E1” (also known as E) and “E2” (also known as E2/NS1). (See, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including E1 and E2.) E1 is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 kDa. By contrast, E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species (Grakoui et al., J. Virol. (1993) 67:1385-1395; Tomei et al., J. Virol. (1993) 67:4017-4026.). The HCV E1 and E2 glycoproteins are of considerable interest because they have been shown to be protective in primate studies. (Choo et al., Proc. Natl. Acad. Sci. USA (1994) 91:1294-1298).
Full-length E1 and E2 are retained within cells and have been shown to lack complex carbohydrate when expressed stably or in a transient Vaccinia virus system (Spaete et al., Virology (1992) 188:819-830; Ralston et al., J. Virol. (1993) 67:6753-6761). Since the E1 and E2 proteins are normally membrane-bound in these expression systems, experimenters had previously thought it desirable to produce secreted forms to facilitate purification of the proteins for further use.
For example, an HCV E2 molecule, truncated at amino acid 661 and which is secreted from mammalian cells, has been described. Spaete et al., Virology (1992) 188:819-830. The production of truncated, secreted HCV E1 and E2 molecules has also been disclosed in International Publication No. WO 96/04301, published Feb. 15, 1996. Inudoh et al., Vaccine (1996) 14:1590-1596, describes the production of an HCV E2 molecule lacking the C-terminal hydrophobic domain. This protein was secreted into culture medium and found to be more antigenic than intracellularly produced counterparts.
Depending on the expression system used, such secreted proteins may not retain the native conformation and may include modified glycosylation patterns. Thus, purification of intracellularly produced HCV E1 and E2 proteins has been attempted in order to preserve the native conformation of the proteins. See, e.g., International Publication No. WO 92/08734, published May 29, 1992.
Despite the above attempts at obtaining HCV E1 and E2, a need still exists for alternative methods of efficiently purifying immunogenic HCV E1 and E2 molecules for use in vaccine compositions and as diagnostic reagents.